Increased ANG II sensitivity following recovery from acute kidney injury: role of oxidant stress in skeletal muscle resistance arteries

Abstract

Ischemia-reperfusion (I/R)-induced acute kidney injury (AKI) results in prolonged impairment of peripheral (i.e., nonrenal) vascular function since skeletal muscle resistance arteries derived from rats 5 wk post-I/R injury, show enhanced responses to ANG II stimulation but not other constrictors. Because vascular superoxide increases ANG II sensitivity, we hypothesized that peripheral responsiveness following recovery from AKI was attributable to vascular oxidant stress. Gracilis arteries (GA) isolated from post-I/R rats (approximately 5 wk recovery) showed significantly greater superoxide levels relative to sham-operated controls, as detected by dihydroeithidium, which was further augmented by acute ANG II stimulation in vitro. Hydrogen peroxide measured by dichlorofluorescein was not affected by ANG II. GA derived from postischemic animals manifested significantly greater constrictor responses in vitro to ANG II than GA from sham-operated controls. The addition of the superoxide scavenging reagent Tempol (10(-5) M) normalized the response to values similar to sham-operated controls. Apocynin (10(-6) M) and endothelial denudation nearly abrogated all ANG II-stimulated constrictor activity in GA from post-AKI rats, suggesting an important role for an endothelial-derived source of peripheral oxidative stress. Apocynin treatment in vivo abrogated GA oxidant stress and attenuated ANG II-induced pressor responses post-AKI. Interestingly, gene expression studies in GA vessels indicated a paradoxical reduction in NADPH oxidase subunit and AT(1)-receptor genes and no effect on several antioxidant genes. Taken together, this study demonstrates that AKI alters peripheral vascular responses by increasing oxidant stress, likely in the endothelium, via an undefined mechanism.

Fig. 1.

Acute kidney injury (AKI) induces alterations in superoxide levels in gracilis arteries (GA). Isolated GA were incubated with dihydroethidium (DHE) and evaluated via fluorescent microscopy. Representative fluorescent images were obtained from vessels derived from sham-operated rats (A and B) and post-AKI rats (C and D). Images were also evaluated prior to (A and C) and following (B and D) stimulation with ANG II (10⁻⁸ M). E: summarized data describing the rate of accumulation of superoxide fluorescence production in arbitrary units (AU) of fluorescent intensity per minute in the presence and absence of ANG II. *Significantly different vs. sham-operated or AKI controls (P < 0.05).

Fig. 2.

AKI induces alterations in hydrogen peroxide levels in GA, which were incubated with dichlorofluoroscein and evaluated via fluorescent microscopy. Representative fluorescent images were obtained from vessels derived from sham-operated rats (A and B) and post-AKI rats (C and D). Images were also evaluated prior to (A and C) and following (B and D) stimulation with ANG II (10⁻⁸ M). E: summarized data describing the rate of accumulation of peroxide production in AU of fluorescent intensity per minute. Values are means ± SE. There were no differences between groups.

Fig. 3.

Responses to ANG II of isolated GA from sham-operated (A) or postischemic (B) rats. GA were subjected to increasing doses of ANG II in the presence and absence of Tempol (n = 7, 10⁻⁴ M). Data are means ± SE and are expressed as absolute change from baseline. There was no effect of Tempol on ANG II responses in sham rats. *P < 0.05 in Tempol treated vs. physiological salt solution control for AKI rats.

Fig. 4.

Effect of AKI on ANG II-induced constriction and the effects of apocynin in isolated GA from postischemic rats. Response to increasing levels of ANG II are shown (white bars) or with increasing levels of ANG II and the NADPH oxidase inhibition with apocynin (1 μM, black bars). Results are shown for vessels obtained from sham-operated controls (A) and post-AKI animals (B). Stippled bars in B represent the restored activity following washout of apocynin, demonstrating restoration of the ANG II response. Data are means ± SE expressed as the absolute change from baseline. *Significant difference from vehicle (n = 4; P < 0.05).

Fig. 5.

Effect of apocynin on norepinephrine-induced constriction in isolated GA from postischemic (AKI) rats. Response to increasing levels of norepinephrine are shown alone (white bars) or with apocynin (1 μM, black bars). Results are shown for vessels obtained from sham-operated controls (A) and postischemic animals (B). Data are means ± SE expressed as the absolute change from baseline. There was no effect of apocynin on norepinepherine responses in either group.

Fig. 6.

Effect of allopurinol on ANG II-induced contriction in isolated GA from postischemic (AKI) rats. Response to increasing levels of ANG II are shown (white bars) or with increasing levels of ANG II and with allopurinol (1 μM, black bars). Allopurinol had no significant effect on the response to ANG II.

Fig. 7.

Effect of endothelial denudation on ANG II-induced constriction in isolated GA from postischemic rats. Response to increasing levels of ANG II are shown in endothelial-denuded vessels obtained from sham-operated controls (black bars) and postischemic animals (white bars). Data are means ± SE expressed as the absolute change from baseline. *Significant difference from sham-operated controls (n = 4, P < 0.05).

Fig. 8.

Effect of apocynin on superoxide fluorescence in skeletal muscle resistance arteries from AKI rats. A: isolated GA were incubated with DHE and evaluated via fluorescent microscopy in the presence and absence of apocynin (10⁻⁵ M) and ANG II (10⁻⁸ M). Results are presented as a ratio from baseline. *Significantly different vs. ANG II (P < 0.05). B: isolated GA were incubated from AKI rats and AKI rats treated with in vivo apocynin in the drinking water (15 mmol/l) between days 28 and 35; DHE fluorescence measurements were conducted at day 35 post-AKI. *Significantly different vs. AKI drinking water only control (n = 4 group, P < 0.05).

Fig. 9.

Effect of apocynin treatment in vivo on acute pressor response to ANG II. Rats were allowed to recover from AKI for 35 days; apocynin treatment occurred on days 28–35 and pressor responses evaluated are based on increasing doses of ANG II as indicated. Data are presented as change in pressure from baseline values. *Significant difference in apocynin-treated vs. nonapocynin-treated AKI by Student's t-test (P < 0.05).